All bacteria possess a single methionine aminopeptidase (MetAP) that is absolutely essential for cell survival. In contrast, eukaryotic cells express two MetAP enzymes and can survive if one of them is eliminated by genetics or inhibition. This enzyme is therefore a potentially attractive target for novel broad- spectrum antibacterial compounds. This key enzyme catalyzes the removal of N-terminal methionine from newly synthesized proteins and requires a divalent metal ion such as Mn(ll), Fe(ll), Co(ll), Ni(ll) orZn(ll) for activity. However, it is currently unclear which of these ions are the most important in vivo. Because most MetAP inhibitors have been discovered and characterized on the Co(ll) enzyme, they may not inhibit other metalloforms of MetAP. The objectives of this proposal are (1) to define the in vivo relevant metalloform of MetAP in E. coli and (2) to identify metalloform-selective MetAP inhibitors as both research tools and leads for drug development. We expect to obtain MetAP inhibitors that are highly selective for each of the relevant metalloforms. These unique inhibitors will be used to identify the in vivo metal utilized by MetAP in E. coli. The successful completion of this research will help to clarify the in vivo metal for MetAP and providenon- peptidic MetAP inhibitors as leads for effective antibacterial agents to treat infectious diseases.